Platelet sarco/endoplasmic reticulum Ca2+ATPase isoform 3b and Rap 1b: interrelation and regulation in physiopathology

Abstract

Platelet Ca2+ signalling involves intracellular Ca2+ pools, whose content is controlled by sarco/endoplasmic reticulum Ca2+ATPases (SERCAs). Among these, a key role is played by the inositol trisphosphate-sensitive Ca2+ pool, associated with the SERCA 3b isoform. We have investigated the control of this Ca2+ pool through the cAMP-dependent phosphorylation of the GTP-binding protein, Rap (Ras-proximate) 1b. We first looked for this Ca2+ pool target of regulation by studying the expression of the different SERCA and Rap 1 proteins in human platelets and various cell lines, by Western blotting and reverse transcription-PCR. Since co-expression of Rap 1b and SERCA 3b was obtained, we looked for their protein-protein interaction as a function of the cAMP-dependent phosphorylation of Rap 1b. Co-immunoprecipitations of SERCA 3b and Rap 1b proteins were found in the absence of phosphorylation, induced by the catalytic subunit of the cAMP-dependent protein kinase (csPKA). In contrast, upon pre-treatment of platelet membranes with csPKA, the SERCA 3b dissociated from the Rap 1b protein, in agreement with a role of its phosphorylated state in their interaction. Finally, we looked for adaptation of this complex in a platelet pathological model of hypertension. We investigated the expression of both proteins, as well as the cAMP-dependent phosphorylation of Rap 1b and SERCA 3b activity in platelets from control normotensive Wistar-Kyoto rats and from spontaneously hypertensive rats (SHRs). A decrease in SERCA 3b activity was associated with a decrease in Rap 1b endogenous phosphorylation in SHR platelets, consistent with a functional role in the regulation of the SERCA 3b-associated Ca2+ pool.

The Small GTPase Rap1 Is Required for Mn2+- and Antibody-induced LFA-1- and VLA-4-mediated Cell Adhesion

“…This result is compatible with recent evidence that extracellular stimuli inducing T lymphocyte adhesion via LFA-1, such as T cell receptor stimulation and phorbol esters, do so by inducing an influx of extracellular calcium (28). Although in several studies Rap1 has been implicated in the regulation of cellular calcium levels (35)(36)(37), clear evidence is still lacking. A recent study demonstrated that Rap2b via interaction with phospholipase C⑀ is able to regulate intracellular calcium signaling in HEK293 cells (38).…”

Cyclic AMP Raises Intracellular Ca 2+ in Human Megakaryocytes Independent of Protein Kinase A

“…These findings are in correspondence with the hypothesis that Rap1 is a molecular "switch" that regulates SERCA activity: the cAMP-induced Rap1 activation is correlated with the inhibition of SERCA and a rise in [Ca 2ϩ ] i (present data), and the PKA-mediated phosphorylation of Rap1 relieves the inhibition, resulting in activation of Ca 2ϩ -ATPases and a decrease in [Ca 2ϩ ] i . 10,11 The latter property is also observed in Ca 2ϩ regulation by phospholamban in cardiac and muscle cells. 23,24 Phosphorylation of this 24-kDa protein by PKA triggers stimulation of cardiac sarcoplasmic reticulum Ca 2ϩ -ATPases, leading to a fall in [Ca 2ϩ ] i .…”

“…Two types of cAMP GEF for Rap1 have been reported: cAMP-GEF-I, also called exchange protein, directly activated by cAMP (Epac), and cAMP-GEF-II. Interestingly, earlier work by Corvazier et al 11 and LacabaratzPorret et al 10 indicated that Rap1 might be involved in Ca 2ϩ regulation in platelets. First, proteins in crude platelet plasma membrane vesicles, ranging in molecular mass from 22 to 29 kDa, bound GTP␥S.…”

“…10,11 Because the guanine nucleotide exchange factor (GEF) for Rap1, Epac1/2, is a target for PKA-independent signaling by cAMP, Rap1 could be the link between cAMP and Ca 2ϩ . 12,13 Using a pulldown technique based on the specific binding of active, GTP-bound Rap1 to the Rap-binding domain of RalGDS, we investigated the effect of iloprost and forskolin on Rap1 activation.…”

The Ryanodine Receptor Calcium Channel of β-Cells

“…In this mode, cAMP promotes Ca 2ϩ release through RY receptor as a consequence of increased filling of the ER by a mechanism that involves cAMP-regulated guanine nucleotide exchange factor and its interaction with Rap1b (26,69). There is no consensus as to the consequences of phosphorylation of IP 3 Rs in terms of Ca 2ϩ release.…”